Hepatitis C virus (HCV) causes infection of humans leading to chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Currently there is no available vaccine for HCV and current treatment of ribavarin-interferon alpha combination is only effective in a fraction of infected individuals. Recent results from our laboratory have led to development of a high resolution, quantitative, genome scale mutational analysis system to study HCV cis-acting elements that are essential for JFH1 (type 2) HCV replication in cell culture. A number of insertions within the HCV IRES (5'UTR) identified in this study were found to increase significantly (~50 fold over wt) the yield of infectious virus in Huh-7.5.1 cells. Additional studies from our laboratory have identified RNA secondary structures/sequences within the HCV 5'UTR that appear to play important role in production of infectious virus. Small insertions with this area lead to production of infectious virus by as much as 100-500 fold compared to the wt. Preliminary studies showed that viral protein synthesis, protein processing and RNA synthesis were similar between the mutants and wt virus. The IRES activity of the mutant 5'UTRs also remained unchanged compared with the wt 5'UTR. Considering the location and context of the mutation (within the highly structured 5'UTR) and the fact that the packaging signal for many viruses are located within the 5'UTR, it is logical to assume that these mutations lead to efficient packaging/assembly of viral RNA. This proposal will utilize genetic, biochemical, and immunological approaches to examine the hypothesis that mutations within defined regions of the JFH1 HCV 5'UTR enhance/reduce RNA packaging/assembly/release. Successful completion of the proposed experiments should identify HCV RNA structures/sequences required for efficient assembly and release of infectious virus particles as well as provide the basis for development of therapeutic antiviral agents that interfere with virus assembly. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) causes infection of humans leading to chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (liver cancer). Currently there is no available vaccine for HCV and current treatment of ribavarin-interferon alpha combination is only effective in a fraction of infected individuals. This proposal will examine how the individual viral components (viral genetic material and viral proteins) assemble to form infectious virus particles in cultured liver cells. The knowledge gained from this study should help develop novel antiviral therapeutic strategies to interfere with the virus assembly process.